The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues. Towards this goal, the laboratory concentrates on the use of screening approaches in proteomics and post-translational modifications. The following major findings were made over the last year: 1)Two projects have been dedicated to monitoring the activity of the Complexes of oxidative phosphorylation (COP) to evaluate the role of post-translational modifications in regulating mitochondrial function. 1a) We have developed an imaging system that permits the kinetic monitoring of COP activity within blue native gel resolved COP in vitro. These studies revealed for the first time that many of these in gel assays are not linear and require sophisticated image processing methods to assure artifacts are avoided. 1b) We have continued to develop a non-destructive optical spectroscopy method using a center mounted integrating sphere and a rapid scanning spectroscopy system to monitor the redox sensitive chromophores of mitochondrial oxidative phosphorylation minimizing light scattering effects. This permits the determination of the activity, or conductance, of numerous steps within the metabolic network generating ATP in the intact mitochondria in near real time. Using this approach we have established characterized all of the redox chromophores in the mitochondria and begun to establish the regulation of reducing equivalent distribution within the network. Initial results have revealed novel redox states within cytochrome oxidase in response to ATP production that suggests a new regulatory site in the regulation of energy metabolism. 2) We have established the role of calcium in regulating the rate of ATP production in skeletal muscle mitochondria. Using some of the methods outlined above, we have surprisingly found that the activation of ATP production in mitochondria is occurring at mutiple levels within the oxidative phosphosphorylation network. In contrast to previous theories, the major impact of calcium is not in the activation of dehydrogenases but the alteration in reaction kinetics at cytochrome oxidase and the F1-F0-ATPase. These data suggest that the regulation of energy conversion in the cell is highly distributed and not controlled by a few rate limiting steps as previous dogma would imply. 3) The location and dynamics of mitochondria within a cell is believed to play an important role in the programming of mitochondrial function along with the protein that is inserted. Working with the investigators from NICHD we have developed a transgenetic mouse line that has a photo convertible fluorescent protein inside the mitochondrial. This protein normal fluorescence occurs in the green, however once it has been treated with UV light it converts to a red fluorescence. This permits labeling of specific pools within an animal and monitoring the movement and potential fusion events in vivo. The initial studies with this probe suggest that mitochondrial motions within differentiated cells, in vivo, might be much slower than those observed in dividing cells in culture. The imaging methods to conduct these studies in vivo are being developed in another project within the laboratory.